Osteoactivin protein and nucleic acids encoding the same, compositions and methods of stimulating bone differentiation

ABSTRACT

The invention provides osteoactivin proteins and nucleic acid molecules that encode the same, as well as biologically functional expression vectors containing nucleic acid molecules encoding osteoactivin proteins, and antibodies specific for osteoactivin proteins. The invention also provides therapeutic and diagnostic compositions and methods for utilizing the proteins, antibodies, nucleic acids, and vectors of the invention, for example, to modulate bone formation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.10/978,758, filed on Nov. 1, 2004, now abandoned, which is a divisionalof U.S. application Ser. No. 09/943,075, filed Aug. 30, 2001, now U.S.Pat. No. 6,812,002, which claims benefit of U.S. Provisional ApplicationNo. 60/229,006, filed Aug. 30, 2000, all hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to the identification of an isolated,full-length rat nucleic acid molecule encoding an osteoactivin protein,therapeutic compositions comprising an osteoactivin protein, and methodsfor using the nucleic acid molecules and proteins for stimulating bonedifferentiation. The invention also relates to methods for treating bonedisorders, including osteopetrosis and osteoporosis.

BACKGROUND OF THE INVENTION

The formation and maintenance of the vertebrate skeleton requires theinteractions of many cell types and growth factors and other molecules.The past decade has witnessed an explosive growth in the generalunderstanding of growth factors and other proteins that mediate thecomplex coordination of bone formation and bone resorption by thesedifferent cell types in skeletal modeling and remodeling (Popoff andMarks, Oral and Maxillofacial Clinics of North America 9:563-579(1997)).

In general, the bone remodeling cycle involves a complex series ofsequential steps that are highly regulated. The initial “activation”phase of bone remodeling begins early in fetal life and is dependent onthe effects of local and systemic growth factors on mesenchymal cells ofthe osteoblast lineage (Eriksen, Endocrinol. Rev. 7:379-408 (1986)).These cells interact with hematopoietic precursors to form osteoclastsin the “resorption” phase. This leads to the differentiation, migrationand fusion of the large multinucleated osteoclasts. These cells attachto the mineralized bone surface and initiate resorption by the secretionof hydrogen ions and lysosomal enzymes. Osteoclastic resorption producesirregular scalloped cavities on bone surface. Once the osteoclasts havecompleted their work of bone removal, there is a “reversal” phase duringwhich mononuclear cells, which may be of the macrophage lineage, arepresent on the bone surface. These cells further degrade collagen,deposit proteoglycans, and release growth factors that signal theinitiation of the “formation” phase. During the final formation phase ofthe remodeling cycle, the cavity created by resorption can be completelyfilled in with successive layers of osteoblasts, which differentiatefrom their mesenchymal precursors and lay down a mineralizable matrix.(Raisz, Clin. Chem. 45:1353-1358 (1999)).

With bone disorders associated with decreased bone mass, osteoclasticresorption outweighs osteoblastic bone formation, resulting in boneloss. While treatments that stimulate bone formation would be beneficialin treating or preventing bone loss, current therapies are suboptimal(Canalis, J. Clin. Invest. 106:177-179 (2000); Raisz, J. Bone Min. Metab17:79-89 (1999)).

An animal model useful in bone studies is the osteopetrosis (op)mutation in the rat. Osteopetrosis describes a group of congenital bonedisorders that are characterized by a generalized increase in skeletalmass resulting from a primary defect in osteoclast-mediated boneresorption (Popoff and Schneider, Molec. Med. Today 2:349-358 (1996)).Numerous osteopetrotic mutations have been described in other species,including human and mouse. The bone that is formed as the skeletondevelops and grows in animals with this mutation is not resorbed,resulting in the failure to develop bone marrow cavities. Theosteopetrotic mutations are pathogenetically heterogeneous since thepoint at which osteoclast development or activation is intercepteddiffers for each mutation (Popoff and Marks, Bone 17:437-445 (1995)).Although osteoclast hypofunction is universal among the osteopetroticmutations, genetic abnormalities involving osteoblastdevelopment/function (i.e., bone formation), mineral homeostasis and theimmune and endocrine systems have also been reported within thisdisorder (Seifert et al., Clin. Orthop. 294:23-33 (1993)).

To date, pharmaceutical approaches to managing osteoporosis orosteopetrosis are of limited effectiveness. Therefore, alternativetherapies are needed to modulate bone cell differentiation and boneformation, and to treat bone disorders such as osteoporosis andosteopetrosis.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery of a novel ratgene encoding an osteoactivin protein. The nucleotide sequence offull-length cDNA of the gene is shown in SEQ ID NO:1. The nucleotidesequence of the cDNA encoding the osteoactivin protein is shown innucleotides 115 to 1,830 of SEQ ID NO:1 and the corresponding amino acidsequence of the osteoactivin protein is shown in SEQ ID NO:2. Thepolynucleotide sequence of the cDNA encoding the osteoactivin proteinlacking the signal sequence is shown in nucleotides 181-1830 of SEQ IDNO:1 and the corresponding osteoactivin polypeptide lacking the signalsequence is from amino acid residues 23-572 of SEQ ID NO:2. The claimedinvention also relates to antibodies which recognize one or moreepitopes of the osteoactivin protein. The claimed invention providestherapeutic compositions comprising (i) a nucleic acid molecule encodingan osteoactivin protein, (ii) an osteoactivin protein, or (iii) anantibody to an osteoactivin protein. These therapeutic compositions areuseful to treat bone disorders and to stimulate bone formation and bonecell differentiation.

Accordingly, in one aspect, the invention is directed to moleculesencoding an osteoactivin protein. One embodiment of this aspect is anucleic acid molecule encoding a rat osteoactivin protein having amolecular weight of 63.8 kilodaltons (kD), wherein said osteoactivinprotein stimulates bone cell differentiation. In a related embodiment ofthis aspect, the invention encompasses a full-length nucleic acidmolecule which encodes an osteoactivin protein, wherein said nucleicacid comprises the nucleic acid sequence of SEQ ID NO:1. In anotherembodiment, the invention provides a nucleic acid molecule encoding anosteoactivin protein, wherein said nucleic acid hybridizes to thecomplement of SEQ ID NO:1 under moderately stringent conditions. In apreferred embodiment, the nucleic acid molecule encoding an osteoactivinprotein having at least 92% sequence identity with the nucleic acidsequence of SEQ ID NO:1 is described. In some embodiments, the nucleicacid molecule encodes a polypeptide comprising SEQ ID NO:2. Theinvention also embodies the nucleic acid molecule encoding anosteoactivin polypeptide comprising amino acid residues 23-572 of SEQ IDNO:2. In other embodiments, the invention provides a nucleic acidencoding an osteoactivin protein, wherein said nucleic acid comprisesfrom nucleotide 115 to nucleotide 1,830 of SEQ ID NO:1. Otherembodiments of the invention provide a polynucleotide encoding anosteoactivin protein lacking the leader sequence, wherein saidpolynucleotide comprises from nucleic acid residues from 181 to 1830 ofSEQ ID NO:1. In still other embodiments, the invention provides anucleic acid encoding an osteoactivin protein, wherein said nucleic acidmolecule hybridizes to the complement of nucleotide 115 to nucleotide1,830 of SEQ ID NO:1 under moderately stringent conditions. In yetanother embodiment of this aspect, the invention further provides anucleic acid molecule encoding an osteoactivin protein having at least92% sequence identity with the nucleic acid sequence from nucleotide 115to nucleotide 1,830 of SEQ ID NO:1.

As used herein, the term “nucleic acid molecule” includes DNA molecules(e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) andanalogs of the DNA or RNA generated, e.g., by the use of nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA. Nucleic acidmolecules include naturally occurring nucleic acid molecules which areseparated from other molecules which are present in the natural sourceof the nucleic acid. For example, a nucleic acid molecule includesgenomic DNA which is separated from the chromosome with which thegenomic DNA is naturally associated. Preferably, a naturally occurringnucleic acid molecule is free of sequences which naturally flank thenucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatednucleic acid molecule can contain less than 5 kilobases (kb), 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequenceswhich naturally flank the nucleic acid molecule in genomic DNA of thecell from which the nucleic acid is derived. Moreover, an isolatednucleic acid molecule, such as a cDNA molecule, is substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

As used herein, the term “osteoactivin protein” refers to a proteinincluding the amino acid sequence of SEQ ID NO:2, the murineosteoactivin protein homolog, nmb, of SEQ ID NO:5, the humanosteoactivin protein homolog, nmb, of SEQ ID NO:6, and the amino acidsequence comprising amino acid residues 23-572 of SEQ ID NO:2. Further,an osteoactivin protein has at least 50% sequence identity, preferably70% sequence identity, and more preferably 90% sequence identity to theamino acid sequence of SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:6, andstimulates bone cell differentiation or bone formation. Preferably, theosteoactivin protein is naturally occurring in a mammalian species.

As used herein, “stimulates bone cell differentiation” means anyincrease in bone cell number or size, including without limitation, theincrease in the rate of bone cell division or precursor bone cellrecruitment from the stem cells or bone marrow cells, and an increase inbone cell size. Such bone cell differentiation can be measured by wellknown cell proliferation assays (e.g., ³H-thymidine incorporation) andbone differentiation assays (e.g., Owen, et al., J. Cell Physiol.143:420-30 (1990)).

As used herein, by “stimulates bone formation” is meant the recruitmentof osteoblasts or osteoblast precursors to a bone site, which results indifferentiation of the cells into mature osteoblasts and their secretionof collagenous matrix which mineralizes into bone matter and increasesbone mass at the site. This term also encompasses the increasedproduction and secretion of collagenous matrix by mature osteoblasts.

As used herein, the term “hybridizes under moderately stringentconditions” describes conditions for hybridization and washing.Stringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. An example of moderately stringent hybridizationconditions is hybridization in 50% formamide 6×SSC at 42° C., followedby one or more washes in 0.2×SSC, 0.1% sodium dodecyl sulfate (SDS) at55° C. In some preferable embodiments, an isolated nucleic acid moleculeof the invention that hybridizes under moderately stringent conditionsto the sequence of SEQ ID NO:1 corresponds to a naturally-occurringnucleic acid molecule.

By two nucleic acid molecules being “complementary” to one another orhybridizing to a “complement” of another nucleic acid molecule is meantthat the first nucleic acid molecule (e.g., an oligonucleotide) is ableto form Watson-Crick base pair hydrogen bonds (i.e., hybridize) with thesecond nucleic acid molecule to form a duplex.

As used herein, a percent “sequence identity” refers to a calculation of“homology” or “identity” between two different nucleic acid or aminoacid sequences (the terms are used interchangeably herein) when thesequences are aligned and compared. The percent sequence identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences, taking into account the number ofgaps, and the length of each gap, which need to be introduced foroptimal alignment of the two sequences.

In another aspect of the invention, the invention features an isolatedand substantially pure osteoactivin protein, or polypeptide fragmentthereof. One preferred embodiment of this aspect of the invention is anisolated and substantially pure rat osteoactivin protein, or polypeptidefragment thereof, wherein said protein comprises the amino acid sequenceof SEQ ID NO:2. In another embodiment, the invention provides anisolated and substantially pure, non-human, non-murine osteoactivinprotein, or polypeptide fragment thereof, having at least 90% sequenceidentity with the amino acid sequence of SEQ ID NO:2, wherein saidosteoactivin protein or polypeptide fragment thereof stimulates bonecell differentiation or bone formation.

An “isolated” or “purified” osteoactivin protein or polypeptide issubstantially free of cellular material or other contaminating proteinsfrom the cell or tissue source from which the protein is derived, orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. In one embodiment, the language “substantiallyfree” means preparation of osteoactivin protein having less than 30%,20%, 10% and more preferably less than 5% (by weight), ofnon-osteoactivin protein (also referred to herein as a “contaminatingprotein”), or of chemical precursors or non-osteoactivin compounds. Whenthe osteoactivin protein, or biologically active portion thereof, isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., the culture medium represents less than 20%, morepreferably less than 10%, and most preferably less than 5% of the volumeof the protein preparation. The invention includes isolated or purifiedpreparations of at least 0.01 milligrams, at least 0.1 milligrams, atleast 1.0 milligrams, and at least 10 milligrams by weight.

Also included, in another aspect of the invention, are expressionvectors containing nucleic acid molecules encoding an osteoactivinprotein or polypeptide fragment therein. In one embodiment, theinvention features a biologically functional expression vectorcomprising a nucleic acid sequence encoding an osteoactivin protein, orbiologically active polypeptide fragment thereof, wherein saidosteoactivin protein comprises the amino acid sequence of SEQ ID NO:2,or has at least 90% sequence identity to the amino acid sequence of SEQID NO:2, and which stimulates bone cell differentiation or boneformation.

In another embodiment of this aspect, the invention is directed tobiologically functional expression vectors comprising a nucleic acidmolecule encoding a rat osteoactivin protein having a molecular weightof 63.8 kD, wherein said osteoactivin protein stimulates bone celldifferentiation. In another embodiment, a biologically functionalexpression vector is provided which comprises the nucleic acid sequenceof SEQ ID NO:1. The invention also encompasses a biologically functionalexpression vector comprising said nucleic acid molecule encoding anosteoactivin protein, wherein the nucleic acid molecule hybridizes tothe complement of SEQ ID NO:1 under moderately stringent conditions. Theinvention also provides a biologically functional expression vectorcomprising a nucleic acid molecule encoding an osteoactivin protein andhaving at least 92% sequence identity with the nucleic acid sequence ofSEQ ID NO:1. In another embodiment, the invention provides abiologically functional expression vector comprising a nucleic acidmolecule encoding an osteoactivin protein, wherein said nucleic acidmolecule comprises from nucleotide 115 to nucleotide 1,830 of SEQ IDNO:1. Yet another embodiment of this aspect of the invention includes abiologically functional expression vector comprising a nucleic acidmolecule encoding an osteoactivin protein, wherein said nucleic acidmolecule hybridizes to the complement of nucleotide 115 to nucleotide1,830 of SEQ ID NO:1 under moderately stringent conditions. Still yetanother embodiment is directed to a biologically functional expressionvector comprising said nucleic acid molecule encoding an osteoactivinprotein having at least 92% sequence identity with the nucleic acidsequence from nucleotide 115 to nucleotide 1,830 of SEQ ID NO:1. In eachof these embodiments, the vector may be a plasmid or a viral vector.

As used herein, the term “vector” refers to a composition capable ofcarrying a nucleic acid molecule to its target. Vectors includeliposomes and nucleic acid molecules capable of transporting anothernucleic acid to which it has been linked. Such nucleic acid vectorsinclude plasmids, cosmids, or viral vectors. The nucleic acid vector canbe capable of autonomous replication or it can integrate into a hostDNA. Viral vectors include, e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses. A “biologically functionalexpression vector” as used herein refers to a vector used to incorporatenucleic acid molecules of the invention, including anosteoactivin-encoding nucleic acid, in a form suitable for expression ina host cell.

In yet another aspect, the invention features immunoglobulins such asantibodies and antigen-binding fragments thereof, that recognize andbind one or more epitopes of the osteoactivin proteins or polypeptidefragments thereof. In one embodiment of this aspect, the inventionprovides a substantially pure antibody that specifically binds to one ormore epitopes of an osteoactivin protein, or a polypeptide fragmentthereof, wherein said osteoactivin protein stimulates bone celldifferentiation. The invention further provides a substantially pureantibody that specifically binds to one or more epitopes of anosteoactivin protein, or a polypeptide fragment thereof, wherein saidosteoactivin protein comprises the amino acid sequence of SEQ ID NO:6.In a related embodiment, a substantially pure antibody that specificallybinds to one or more epitopes of an osteoactivin protein, or polypeptidefragment thereof, wherein said osteoactivin protein comprises the aminoacid sequence of SEQ ID NO:2 is provided. In a preferred embodiment, theantibody is selected from the group consisting of an antibody whichbinds to one or more epitopes of an osteoactivin peptide 35 having SEQID NO:3 and an antibody which binds to one or more epitopes of anosteoactivin peptide 551 having SEQ ID NO:4. In a particularly preferredembodiment, the antibody binds to one or more epitopes of amino acids538-553 of SEQ ID NO:6. Another preferred embodiment is an antibodywhich specifically binds to an osteoactivin protein, or polypeptidefragment thereof, having at least 90% sequence identity with the aminoacid sequence of SEQ ID NO:2, wherein said osteoactivin protein orpolypeptide fragment thereof stimulates bone cell differentiation orbone formation. In each of these embodiments, the antibody may be apolyclonal or a monoclonal antibody.

The term “antibody” as used herein refers to an immunoglobulin moleculeor immunologically active portion thereof, i.e., an antigen-bindingportion. Examples of immunologically active portions of immunoglobulinmolecules include F(ab), Fv, and F(ab′)₂ fragments which can begenerated by cleaving the antibody with an enzyme such as pepsin.

The term “epitope” as used herein means that region of amino acidresidues of an osteoactivin protein antigen that is specificallyrecognized by an anti-osteoactivin antibody.

By “specifically binds” means an antibody that physically interacts withits specific ligand (i.e., an osteoactivin protein or biologicallyactive polypeptide fragment thereof) with greater affinity that it bindsto other molecules.

The invention further provides methods for producing a substantiallypure osteoactivin protein, or polypeptide fragment thereof, comprising:(a) culturing a cell stably transformed with a gene comprising a nucleicacid molecule encoding an osteoactivin protein, wherein said nucleicacid comprises the nucleic acid sequence from nucleotide 115 tonucleotide 1,830 of SEQ ID NO:1; and (b) isolating and purifying theosteoactivin protein from the culture medium. Another preferredembodiment includes a method for producing a substantially pureosteoactivin protein, or polypeptide fragment thereof, comprising: (a)culturing a cell stably transformed with a gene comprising said nucleicacid molecule encoding an osteoactivin protein having at least 92%sequence identity with the nucleic acid sequence from nucleotide 115 tonucleotide 1,830 of SEQ ID NO:1; and (b) isolating and purifying saidosteoactivin protein from said culture medium. A method for producing asubstantially pure osteoactivin protein, or polypeptide fragmentthereof, comprising: (a) culturing a cell stably transfected with avector comprising the nucleic acid molecule encoding an osteoactivinprotein, wherein said nucleic acid comprises the nucleic acid sequencefrom nucleotide 115 to nucleotide 1,830 of SEQ ID NO:1; and (b)isolating and purifying said osteoactivin protein from said culturemedium, is also provided. In a related embodiment, the inventionprovides a method for producing a substantially pure osteoactivinprotein, or polypeptide fragment thereof, comprising: (a) culturing acell stably transfected with a vector comprising said nucleic acidmolecule encoding an osteoactivin protein having at least 92% sequenceidentity with the nucleic acid sequence from nucleotide 115 tonucleotide 1,830 of SEQ ID NO:1; and (b) isolating and purifying saidosteoactivin protein from said culture medium.

As used herein, the terms osteoactivin “gene” and “recombinant gene”refer to nucleic acid molecules which include an open reading frameencoding an osteoactivin protein, such as a mammalian osteoactivinprotein, and can further include non-coding regulatory sequences, andintrons. These genes can be isolated from genomic DNA, cloned byrecombinant means, or chemically synthesized.

As used herein, the terms “transformation” and “transfection” areintended to refer to a variety of art-recognized techniques forintroducing foreign nucleic acid (e.g., DNA) into a prokaryotic oreukaryotic host cell, including, but not limited to, calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation, such that the DNA withinthe vector is expressed in the host cell.

The invention also provides for therapeutic compositions of thedisclosed nucleic acid molecules and osteoactivin proteins andantibodies. Accordingly, in another aspect, the invention provides atherapeutic composition comprising a pharmaceutically acceptable carrieror delivery vehicle and a nucleic acid encoding an osteoactivin protein,or biologically active polypeptide fragment thereof, wherein saidosteoactivin protein stimulates bone cell differentiation. In someembodiments, the therapeutic composition comprises a nucleic acidmolecule encoding a human osteoactivin or encoding the amino acidsequence of SEQ ID NO:6. In still another embodiment, the inventionencompasses a therapeutic composition comprising a nucleic acid moleculeencoding an osteoactivin protein having at least 92% sequence identitywith the nucleic acid sequence of SEQ ID NO:1, and a pharmaceuticallyacceptable delivery vehicle.

In certain embodiments of this aspect of the invention, the therapeuticcomposition further comprises a mediator such as a cytokine or a growthfactor. Non-limiting examples of such mediators include interleukin-1,tumor necrosis factor, lymphotoxin, interleukin-6, prostaglandins of theE-series, leukotrienes, lipopolysaccharides, transforming growthfactor-β, and colony-stimulating factors. In another related aspect, theinvention provides a therapeutic composition comprising an agent thatstimulates osteoactivin-mediated bone formation. The term “mediator”refers to a molecule that directly modulates, mediates, or changes theexpression of an osteoactivin protein.

As used herein, a “therapeutic composition” refers to a compositioncomprising an active ingredient required to cause a desired effect whena therapeutically effective amount of the composition is administered toa mammal in need thereof.

Within the present invention, a “therapeutically effective amount” of acomposition is that amount of each active component of the therapeuticcomposition that is sufficient to show a benefit (e.g., a reduction in asymptom associated with the disorder, disease, or condition beingtreated). When applied to an individual active ingredient, administeredalone, the term refers to that ingredient alone. When applied to acombination, the term refers to combined amounts of the activeingredients that result in the benefit, whether administered incombination, serially, or simultaneously.

As used herein, the term “pharmaceutically acceptable delivery vehicle”refers to carriers that do not negatively affect the biological activityof the therapeutic molecule or compound to be placed therein.Preferably, the vehicle targets bone cells. The characteristics of thedelivery vehicle will depend on the route of administration. Therapeuticcompositions may contain, in addition to the active compound, diluents,fillers, salts, buffers, stabilizers, solubilizers, and other materialswell known in the art.

Other therapeutic compositions within the scope of this invention covercompositions comprising vectors. Accordingly, in one embodiment, theinvention features a therapeutic composition comprising a biologicallyfunctional expression vector comprising a nucleic acid sequence encodingan osteoactivin protein, wherein said osteoactivin protein stimulatesbone cell differentiation or bone formation, and a pharmaceuticallyacceptable delivery vehicle. In another embodiment, the inventionprovides a therapeutic composition comprising a biologically functionalexpression vector comprising a nucleic acid molecule encoding anosteoactivin protein having at least 92% sequence identity with thenucleic acid sequence of SEQ ID NO:1, and a pharmaceutically acceptabledelivery vehicle. In certain embodiments of this aspect of theinvention, the therapeutic composition further comprises a mediator,such as interleukin-1, tumor necrosis factor, lymphotoxin,interleukin-6, prostaglandins of the E-series, leukotrienes,lipopolysaccharides, transforming growth factor-β, or colony-stimulatingfactors, or a nucleic acid molecule encoding any of these mediatorpolypeptides.

Still other embodiments within this aspect of the invention are directedto therapeutic compositions comprising an osteoactivin protein.Accordingly, the invention features a therapeutic composition comprisinga pharmaceutically acceptable carrier or delivery vehicle and anosteoactivin protein, or biologically active polypeptide fragmentthereof, wherein said osteoactivin protein stimulates bone celldifferentiation or bone formation. In some embodiments, the osteoactivinprotein in the therapeutic composition is human. In other embodiments,the osteoactivin protein comprises SEQ ID NO:6. In another embodiment ofthis aspect, the invention covers a therapeutic composition comprisingan osteoactivin protein, or polypeptide fragment thereof, wherein saidprotein comprises the amino acid sequence of SEQ ID NO:2, and apharmaceutically acceptable delivery vehicle. In yet a furtherembodiment, a therapeutic composition comprising a pharmaceuticallyacceptable delivery vehicle and a non-human, non-murine osteoactivinprotein, or polypeptide fragment thereof, having at least 90% sequenceidentity with the amino acid sequence of SEQ ID NO:2, wherein saidosteoactivin protein or polypeptide fragment thereof stimulates bonecell differentiation or bone formation. In certain embodiments of thisaspect of the invention, the therapeutic composition may furthercomprises a mediator, including interleukin-1, tumor necrosis factor,lymphotoxin, interleukin-6, prostaglandins of the E-series,leukotrienes, lipopolysaccharides, transforming growth factor-β, andcolony-stimulating factors. In another aspect, the invention provides atherapeutic composition comprising an agent that inhibitsosteoactivin-mediated bone formation, and a pharmaceutically acceptabledelivery vehicle.

As used herein, a “biologically active portion” of an osteoactivinprotein includes a fragment of an osteoactivin protein which is capableof affecting bone differentiation or bone formation.

Additional therapeutic compositions of the invention relate to thosecomprising antibodies that react with, or specifically bind,osteoactivin proteins. In one embodiment of this aspect, the inventionprovides a therapeutic composition comprising a pharmaceuticallyacceptable delivery vehicle and an antibody that specifically binds toone or more epitopes of an osteoactivin protein, or polypeptide fragmentthereof, wherein said osteoactivin protein stimulates bonedifferentiation or bone formation. In a related embodiment, theinvention covers a therapeutic composition comprising a substantiallypure antibody that specifically binds to one or more epitopes of anosteoactivin protein, or polypeptide fragment thereof, wherein saidosteoactivin protein comprises the amino acid sequence of SEQ ID NO:2,and a pharmaceutically acceptable delivery vehicle. In a preferredembodiment, the antibody of a therapeutic composition is selected fromthe group consisting of an antibody which binds to one or more epitopesof an osteoactivin peptide 35 having SEQ ID NO:3, an antibody whichbinds to one or more epitopes of an osteoactivin peptide 551 having SEQID NO:4, and an antibody which binds to one or more epitopes of aminoacids 538-553 of the human osteoactivin protein of SEQ ID NO:6, togetherwith a pharmaceutically acceptable delivery vehicle. Another preferredantibody, according to this aspect of the invention, is one in which theantibody of the therapeutic composition specifically binds to anon-human, non-murine osteoactivin protein, or polypeptide fragmentthereof, having at least 90% sequence identity with the amino acidsequence of SEQ ID NO:2, wherein said osteoactivin protein orpolypeptide fragment thereof stimulates bone cell differentiation orbone formation, together with a pharmaceutically acceptable deliveryvehicle. In certain embodiments, the antibody of the invention is apolyclonal or a monoclonal antibody.

In yet another aspect, the invention provides in vivo methods ofstimulating bone formation in a mammal, comprising administering to saidmammal a therapeutically effective amount of a nucleic acid moleculeencoding an osteoactivin protein or peptide thereof, or an osteoactivinprotein, or biologically active polypeptide fragment thereof, or anagent that enhances osteoactivin-mediated bone cell differentiation orbone formation. In other embodiments of this aspect, ex vivo methods forstimulating bone formation in a human are described, comprising thesteps of: (a) extracting osteoblast cells from said human; (b)contacting said osteoblast cells with a therapeutically effective amountof a nucleic acid molecule encoding an osteoactivin protein, or anosteoactivin protein, or biologically active polypeptide fragmentthereof; and (c) returning said cells to the bone of said human.

“Mammal” as used herein means any animal classified as a mammalincluding humans, cows, horses, dogs, mice, cats, goats, pigs, andsheep.

In another aspect, the invention features in vivo methods for inhibitingbone formation in a mammal, comprising administering to said mammal atherapeutically effective amount of any of the therapeutic compositionsof the present invention comprising antibodies. In a related aspect, theinvention also provides a method for inhibiting bone formation in amammal, comprising administering to said mammal a therapeuticallyeffective amount of an agent that inhibits osteoactivin-mediated boneformation.

In another aspect, the invention provides in vivo methods of inhibitingbone formation or bone cell differentiation in a mammal, comprisingadministering to said mammal a therapeutically effective amount of anyof the therapeutic compositions of the invention comprising, in part, anantibody.

The invention provides, in yet another aspect, in vivo methods oftreating bone disorders in a mammal, such as a human, comprisingadministering to said mammal a therapeutically effective amount of anyof the therapeutic compositions of the invention, comprising ananti-osteoactivin antibody or an agent that inhibitsosteoactivin-mediated bone differentiation. In a related embodiment, theinvention provides ex vivo methods for treating bone disorders in amammal, comprising the steps of: (a) extracting osteoblast cells fromsaid mammal; (b) contacting said osteoblast cells with a therapeuticallyeffective amount of an antibody specific for osteoactivin protein or anagent that inhibits osteoactivin-mediated bone cell differentiation orbone formation; and (c) returning said contacted cells to the bone ofsaid mammal. In preferred embodiments, the bone disorder treated by themethod is selected from the group consisting of an ectopic boneformation, osteoporosis, periodontal disease, and osteopetrosis.

The phrase “bone disorder,” as used herein, refers to a pathologicaldisorder, disease, or condition in a mammal in which there is animbalance in the ratio of bone formation to bone resorption, such that,if left untreated, would result in that mammal exhibiting an abnormalmass of bone.

“Treating,” “treatment,” and “therapy,” as used herein, refer tocurative, prophylactic, or preventative manipulations, or manipulationswhich stimulate bone cell differentiation or bone formation, postponethe development of bone disorder symptoms, and/or reduce the severity ofbone disorders and/or such symptoms that will or are expected to developfrom a bone disorder. The terms further include ameliorating existingbone disorder symptoms, preventing additional symptoms, ameliorating orpreventing the underlying metabolic causes of symptoms, preventing orreversing metabolic causes of symptoms, preventing or reversing bonegrowth, and/or encouraging bone resorption. Thus, the terms denote thata beneficial result has been conferred on a mammal with a bone disorder,or with the potential to develop such disorder.

In another aspect, the invention provides in vivo methods of treatingbone disorders in a mammal such as a human, comprising administering tosaid mammal, a therapeutically effective amount of any of therapeuticcompositions of the invention comprising a nucleic acid moleculeencoding an osteoactivin protein, or an osteoactivin protein, orbiologically active polypeptide fragment thereof, wherein saidosteoactivin protein or biologically active polypeptide fragment thereofstimulates bone formation or bone cell differentiation. In otherembodiments of this aspect, ex vivo methods for treating bone disordersin a mammal are provided. These methods comprise the steps of: (a)extracting osteoblast cells from said mammal; (b) contacting saidosteoblast cells with a therapeutically effective amount of any of thetherapeutic compositions comprising a nucleic acid molecule encoding anosteoactivin protein, or biologically active polypeptide fragmentthereof, of the invention; and (c) returning said cells to the bone ofsaid mammal, wherein said osteoactivin protein or biologically activepolypeptide fragment thereof stimulates bone formation or bone celldifferentiation. In certain embodiments, the bone disorder treated isselected from the group consisting of an ectopic bone formation,osteoporosis, periodontal disease, and osteopetrosis. In other certainembodiments of this aspect, the method results in inhibition of boneresorption.

In yet another aspect, the invention provides methods for identifying anagent that modulates the expression or activity of osteoactivin nucleicacid molecules or proteins.

As used herein, an “agent” is a candidate or test compound (e.g., aprotein, peptide, peptidomimetic, peptoid, small molecule or otherchemical entity) which modulates the expression or activity of theosteoactivin nucleic acid molecule or protein. By “modulating theexpression or activity of the osteoactivin nucleic acid molecule orprotein” is meant a compound or molecule that has a stimulatory orinhibitory effect on, for example, osteoactivin expression orosteoactivin activity.

The method uses cells capable of expressing a gene under the control ofthe regulatory element(s) of an osteoactivin gene. Such cells includethose which are capable of expressing an endogenous osteoactivin gene(e.g., an osteoblast cell line) or a cell transfected with a transgenecomprising an osteoactivin regulatory element (e.g., an osteoactivinpromoter) fused to a nucleic acid sequence encoding a polypeptide (e.g.,an osteoactivin protein or a reporter protein), such that theosteoactivin gene regulatory element controls expression of the codingsequence.

In a preferred embodiment, the method uses host cells transfected with anucleic acid comprising an osteoactivin regulatory element fused withnucleic acid sequence encoding a reporter protein. The preferredosteoactivin regulatory element comprises sequences spanning from justupstream of the ATG start site to 8-10 kb upstream of the ATG startsite. The method comprises culturing separate samples of cells in thepresence and absence of an agent in a suitable culture medium, whereinsaid cells express a gene under the control of an osteoactivinregulatory element; and measuring and comparing the levels of expressionof said gene from said samples of cells cultured in the presence andabsence of agent.

In still another aspect, the invention provides assays for determiningthe presence or absence of a genetic alteration in an osteoactivinpolypeptide or in a nucleic acid encoding an osteoactivin protein. Oneembodiment of the invention is an assay for diagnosing osteopetrosis ina mammal suspected of suffering from osteopetrosis, comprising: (a)measuring the level of osteoactivin protein expression in a biologicalsample from said mammal; and (b) comparing said level of osteoactivinprotein expression to a level of osteoactivin protein expression in abiological sample from a control.

The term “biological sample” includes tissues, cells, and biologicalfluids isolated from a mammal, as well as tissues, cells and fluidspresent within a mammal.

In another aspect, a method for diagnosis of osteopetrosis in a mammalis provided. In this method, the level of osteoactivin in the mammal ismeasured and compared with the level of osteoactivin expressed in acontrol mammal which does not suffer from osteopetrosis, whereinincreased expression in (a) compared to (b) is indicative ofosteopetrosis in the mammal in (a).

It is to be understood that the foregoing general description and thefollowing detailed description are exemplary and explanatory, and areintended to provide further explanation of the invention claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the nucleotide sequence (SEQ IDNO:1) and corresponding amino acid sequence of rat osteoactivin and itspredicted amino acid sequence (SEQ ID NO:2) (beginning with themethionine at nucleotide 115) shown in single letter format below theDNA sequence. Solid black lines between nucleotides 217 to 267 and 1768to 1818 underline the peptides to which the antisera were raised forimmunohistochemical localization and Western blot analysis ofosteoactivin expression.

FIG. 1B is a chart characterizing the structure of the humanosteoactivin gene from BAC clone RG271G13.

FIG. 1C is a graphic representation of the results of hydropathyanalysis of osteoactivin.

FIG. 2A is a schematic representation of the alignments of the openreading frame nucleotide sequences of rat osteoactivin (SEQ ID NO:1),mouse nmb (SEQ ID NO:7), and human nmb (SEQ ID NO:8).

FIG. 2B is a schematic representation of the alignment of the predictedamino acid sequences of rat osteoactivin (SEQ ID NO:2), mouse nmb (SEQID NO: 5), and human nmb (SEQ ID NO:6).

FIG. 3 is a representation of an autoradiograph of a differentialdisplay gel showing osteoactivin (arrow) in mutant (M) and normal (N)long bone (L) and calvaria (C).

FIG. 4A is a representation of a Northern blot showing osteoactivinexpression in the mutant calvaria (M) which was 5-to 7-fold higher thanin the normal calvaria (N). Similar results were obtained when RNA frommutant (M) and normal (N) long bone were compared.

FIG. 4B is a representation of the same Northern blot shown in FIG. 4Aafter stripping and reprobing with a probe for 18s rRNA.

FIG. 5 is a representation showing the immunolocalization ofosteoactivin in primary rat osteoblasts, wherein immunofluorescentstaining was primarily observed in the perinuclear region of the cell,consistent with localization in the secretory pathways of the cell.

FIG. 5A is a representation of an electron micrograph showing theimmunolocalization of osteoactivin in primary rat osteoblasts, whereinimmunofluorescent staining was primarily observed in the perinuclearregion of the cell, consistent with localization in the secretorypathways of the cell. Primary rat osteoblasts cultured for 5 days werefixed and incubated with chicken anti-osteoactivin primary antibodyfollowed by incubation with a Cy3-conjugated secondary antibody (red).Magnification 175×; insert magnification 350×.

FIG. 5B is a representation of an electron micrograph showing theimmunolocalization of the rough endoplasmic reticular (RER) in primaryrat osteoblasts. Cells were then stained to visualize the RER usingDiOC₅ dye (green). Magnification 175×; insert magnification 350×.

FIG. 5C is a representation of an electron micrograph showing theco-localization of immunofluorescent staining of osteoactivin with theimmunofluorescent staining of the rough endoplasmic reticulum (RER).Magnification 175×; insert magnification 350×. Images of FIG. 5A andFIG. 5B were overlaid to demonstrate co-localization of staining forosteoactivin with the RER (yellow).

FIG. 6 is a representation of a Northern blot of osteoactivin expressionin calvaria and long bone of mutant (M) and normal (N) rats 2 weeks (2wk), 4 weeks (4 wk) and 6 weeks (6 wk) old. Osteoactivin was expressedat higher levels in the mutant bones at all ages examined, and appearedto decrease with age in the normal rats, while in the mutants expressionremained high, especially in the long bone RNA.

FIG. 7A is a representation of a Northern blot showing osteoactivinexpression in primary rat osteoblast cells derived from normal (N) ormutant (M) calvaria cultured for 1 week, 2 weeks, or 3 weeks.

FIG. 7B is a representation of the same Northern blot in FIG. 7A whichwas stripped and reprobed with a probe for 18S rRNA.

FIG. 8 is a representation of a Western blot showing secretedosteoactivin protein from osteoblasts isolated and cultured for one weekfrom either mutant (M) or normal (N) rat calvaria and probed with achicken anti-rat osteoactivin antibody raised against peptide 551 (SEQID NO:4).

FIG. 9 is a graphic representation of the quantitation of a Western blotof osteoactivin expression (normalized as to β-tubulin as a control forprotein loading and blotting efficiency) in long bones from 2 week (2wk), 4 week (4 wk), and 6 week (6 wk) old mutant (M) or normal (N) rats.

FIG. 10 is a graphic representation of the ability of anti-osteoactivinantibodies to inhibit osteoblast differentiation as measured by calciumdeposition.

DETAILED DESCRIPTION OF THE INVENTION

The patent applications, patents, and literature references cited hereinindicate the knowledge of those of ordinary skill in this field and arehereby incorporated by reference in their entirety. In the case ofinconsistencies between any reference cited herein and the specificteachings of the present disclosure, this disclosure will prevail.Similarly, any inconsistencies between an art-understood meaning of aterm and a meaning of a term as specifically taught in the presentdisclosure will be resolved in favor of this disclosure.

The present invention discloses a cDNA encoding a novel protein,osteoactivin, first identified in the bone of rats carrying the opmutation. A comparison of gene expression in normal versus op long bonesand calvaria using mRNA-differential display resulted in theidentification and cloning of a cDNA encoding the osteoactivin protein.Osteoactivin mRNA is highly over-expressed in op versus normal bone.These findings provide evidence that the protein encoded by this cDNAplays a role in osteoblast development, bone cell differentiation, andbone formation, and therefore, is involved in normal skeletalmodeling/remodeling.

Nucleic Acid Molecules Encoding an Osteoactivin Protein

The rat full-length osteoactivin nucleic acid sequence (FIG. 1A; SEQ IDNO:1), which is approximately 2320 nucleotides long includinguntranslated regions, contains a predicted methionine-initiated codingsequence of 1716 nucleotides (nucleotides 115-1830 of SEQ ID NO:l). Thisfull-length nucleic acid sequence has been deposited in GenBank and hasAccession Number AF184983.

A GenBank search identified human and mouse nmb proteins found inmelanoma cell lines that are likely homologs of the rat osteoactivinprotein disclosed herein (Waterman et al., Int. J. Cancer 60:73-81(1995); Bachner et al., GenBank Accession No. AJ 251685). A comparisonof the nucleotide sequences of the open reading frames of the ratosteoactivin, human nmb, and mouse nmb, genes is shown in FIG. 2A. Ofthe 1716 nucleotides in the rat osteoactivin coding region, 1304nucleotides were identical in human, which corresponds to 76% sequenceidentity. Of the 1716 nucleotides in the rat osteoactivin coding region,1574 nucleotides were identical in mouse, which corresponds to 91%sequence identity.

To determine the percent sequence identity of two amino acid sequences,or of two nucleic acid sequences, the sequences were aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). Preferably, the length of a reference sequence aligned forcomparison purposes is at least 30%, more preferably at least 40%, evenmore preferably at least 50%, even more preferably at least 60%, andeven more preferably at least 70% of the length of the referencesequence. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions were then compared. When aposition in the first sequence was occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, the molecules were considered identical at that position. Thepercent sequence identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. For example, the percent sequence identity between two aminoacid sequences is determined using the Needleman and Wunsch algorithm(J. Mol. Biol. 48:444-453 (1970)) which has been incorporated into theGAP program in the GCG software package, using either a Blossum 62matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or4 and a length weight of 1, 2, 3, 4, 5, or 6. Alternatively, the percentsequence identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package, using a NWSgapdna.CMPmatrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and theone that should be used if the practitioner is uncertain what parametersshould be applied to determine if a molecule is within a sequenceidentity or homology limitation of the invention) are a Blossum 62scoring matrix with a gap penalty of 12, a gap extend penalty of 4, anda frameshift gap penalty of 5. The percent sequence identity between twoamino acid or nucleotide sequences can also be determined using thealgorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which hasbeen incorporated into the ALIGN program (version 2.0), using a PAM120weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul et al. (J. Mol. Biol. 215:403-10 (1990)). BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to osteoactivinnucleic acid molecules of the invention. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3 to obtainamino acid sequences homologous to osteoactivin protein molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al. (Nucleic AcidsRes. 25(17):3389-3402 (1997)). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used (see, NCBI website).

A GenBank search using the rat osteoactivin cDNA as the query identifiedthe presence of the human osteoactivin gene on BAC clone RG27G13.Alignment of the human osteoactivin/nmb cDNA sequence with this BACclone demonstrates that the human osteoactivin transcript is encoded by11 exons spanning 28.3 kb, as shown in FIG. 1B. These exons range insize from 95 bp to 1019 bp. Southern blot analysis indicates thatosteoactivin is a single copy gene in the human genome. FISH analysis,radiation hybrid mapping, and bioinformatic localization all place thehuman osteoactivin gene on chromosome ⁷p15.1. No other genes involved inbone metabolism have been reported at this locus. 5′ RACE analysis ofhuman osteoactivin in both human osteoblasts and kidney mRNA demonstratethat the same transcriptional initiation site was used in both tissuesand that this site mapped to the end of the human nmb cDNA as previouslyreported. Osteoactivin is expressed in human osteoblasts in culture as asingle transcript of approximately 2.4 kb.

The invention further contemplates nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1, or the nucleotidesequence of the DNA insert of the plasmid deposited with GenBank asAccession Number AF184983. Such differences can be due to degeneracy ofthe genetic code, and result in a nucleic acid which encodes the sameosteoactivin proteins as those encoded by the nucleotide sequencedisclosed herein. The invention provides an isolated nucleic acidmolecule encoding a protein having an amino acid sequence which differsby at least 1, but by less than 5, 10, 20, 50, or 100 amino acidresidues than shown in SEQ ID NO:2. If alignment is needed for thiscomparison, the sequences should be aligned for maximum homology.“Looped” out sequences from deletions or insertions, or mismatches, areconsidered differences.

Nucleic acid molecules of the invention can be chosen for having codonswhich are preferred or non-preferred for a particular expression system.For example, the nucleic acid can be one in which at least one codon,and preferably at least 10%, or 20% of the codons, have been alteredsuch that the sequence is optimized for expression in, e.g., E. coli,yeast, human, insect, or Chinese hamster ovary (CHO) cells.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologs (different locus), and orthologs(different organism), or can be non-naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product), as describedbelow.

Preferably, the nucleic acid sequence differs from that of SEQ ID NO:1,or the sequence in GenBank Accession Number AF184983, e.g., by at leastone nucleotide but less than 10, 20, 30, or 40 nucleotides.Alternatively, the nucleic acid sequence differs from that of SEQ IDNO:1 or of AF18493 by at least one but less than 1%, 5%, 10% or 20% ofthe nucleotides in the subject nucleic acid. If necessary for thisanalysis the sequences should be aligned for maximum homology. “Looped”out sequences from deletions or insertions, or mismatches, areconsidered differences.

SignalP analysis (CBS SignalP V1.1 World Wide Web Prediction Server atthe Center for Biological Sequence (Technical University of Denmarkwebsite)) was used to analyze the sequence of osteoactivin for thepresence of a signal peptide. Signal P calculates the maximal C-(rawcleavage site score), S-(signal peptide score), and Y-(combined cleavagesite score) scores, and the mean S-score, between the N-terminal and thepredicted cleavage site. SignalP analysis of osteoactivin revealed amean S score of 0.907, indicating that osteoactivin has a signalsequence. The cleavage site for the signal peptide was predicted tooccur between residues 22 and 23 of rat osteoactivin (SEQ ID NO:2). Thisregion is conserved across species, indicating that mouse and humanosteoactivin contain the same leader sequence and cleavage site.Accordingly, osteoactivin polypeptides lacking the signal sequence arefunctionally active osteoactivin polypeptides.

The invention, therefore, provides for nucleic acid molecules encodingthe osteoactivin polypeptide lacking the signal sequence, wherein theosteoactivin polypeptide comprises amino acid residues 23-572 of SEQ IDNO:2. This nucleic acid molecule encoding the osteoactivin proteinlacking the signal sequences comprises the nucleic acid sequence ofnucleotides 181-1830 of SEQ ID NO:1.

Orthologs, homologs, and allelic variants can be identified usingmethods known in the art. These variants comprise a nucleotide sequenceencoding a polypeptide that is 50%/o, at least 55%, typically at least70-75/o, more typically at least 80-85%, and most typically at least90-95% or more identical to the nucleotide sequence shown in SEQ ID NO:1or to a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderatelystringent conditions to the nucleotide sequence shown in SEQ ID NO:1, ora fragment of the sequence. Nucleic acid molecules corresponding toorthologs, homologs, and allelic variants of the osteoactivin cDNAs ofthe invention can further be isolated by mapping to the same chromosomeor locus as the osteoactivin gene. Preferred variants are those thatmaintain their biological function including the ability to bindosteoactivin binding partners.

An “allelic variant,” as used herein, is a protein having at least 75%identity, preferably at least 85%, more preferably at least 95%, andmost preferably at least 99% identity to the amino acid sequence ofosteoactivin, or to a fragment thereof, or to a protein conjugatethereof which retains the biological activity of osteoactivin. Allelicvariants of osteoactivin include both functional and non-functionalproteins. Functional allelic variants are naturally occurring amino acidsequence variants of the osteoactivin protein within a population thatmaintain their biological function including the ability to bindosteoactivin binding partners. Functional allelic variants willtypically contain only conservative substitution(s) of one or more aminoacids of SEQ ID NO:2, or the substitution, deletion or insertion ofnon-critical residues in non-critical regions of the protein.Non-functional allelic variants are naturally-occurring amino acidsequence variants of the osteoactivin protein, e.g., human osteoactivinprotein, within a population that does not have osteoactivin biologicalactivity such as the ability to bind osteoactivin binding partners.Non-functional allelic variants will typically contain anon-conservative substitution, a deletion or insertion, or prematuretruncation of the amino acid sequence of SEQ ID NO:2, or a substitution,insertion, or deletion in critical residues or critical regions of theprotein.

Osteoactivin Proteins and Polypeptide Fragments

The nucleotide coding sequence encodes a 572 amino acid protein shown inFIG. 1A (SEQ ID NO:2). The protein has a predicted molecular weight of63.8 kD. Hydropathy analysis, shown in FIG. 1C, reveals a potentialleader sequence with a cleavage site after amino acid residue 22 in SEQID NO:2, as well as several potential transmembrane spanning regionsthroughout the molecule.

FIG. 2B shows the amino acid sequences of rat osteoactivin, human nmb,and mouse nmb, aligned to determine the percentage of sequence identity.Of the 572 amino acid residues in the rat osteoactivin protein, 394amino acid residues were identical in human, which corresponds to 69%sequence identity. Notably, the predicted protein sequence of the ratosteoactivin has a proline/serine-rich 14 amino acid residue insertionbeginning at amino acid residue 33 that is not present in the human nmbhomolog. Of the 572 amino acid residues in the rat osteoactivin protein,509 amino acid residues were identical to mouse, which corresponds to89% sequence identity.

An osteoactivin protein of the invention is a protein which comprisesthe amino acid sequence of SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:6, andalternatively or additionally, comprises an osteoactivin protein havingat least 90% sequence identity to the amino acid sequence of SEQ IDNO:2, SEQ ID NO:5 or SEQ ID NO:6 and stimulates bone celldifferentiation or bone formation. An osteoactivin protein of theinvention further comprises the osteoactivin protein sequence lackingthe signal sequence comprising amino acid residues 23-572 of SEQ IDNO:2.

The osteoactivin proteins, polypeptide fragments thereof, mutants,truncations, derivatives, and splice variants of SEQ ID NO:2 thatdisplay substantially equivalent or altered osteoacfivin activityrelative to SEQ ID NO:2 are likewise contemplated. These variants may bedeliberate, for example, such as modifications obtained throughsite-directed mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the osteoactivinprotein. Included within the scope of these terms are osteoactivinproteins specifically recited herein, as well as all substantiallyhomologous analogs and allelic variants.

Analogs may be made through substitution of conserved amino acids. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted non-essential amino acid residue in an osteoactivin protein ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of an osteoactivin codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for osteoactivin biological activity to identify mutantsthat retain activity. Following mutagenesis of SEQ ID NO:1, or thenucleotide sequence of the DNA insert of the plasmid deposited withGenBank as Accession Number AF184983, the encoded protein can beexpressed recombinantly and the activity of the protein can bedetermined.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of osteoactivin (e.g., the sequence of SEQID NO:1, or the nucleotide sequence of the DNA insert of the plasmiddeposited with GenBank as Accession Number AF184983) without abolishingor, more preferably, without substantially altering a biologicalactivity, whereas an “essential” amino acid residue results in such achange. For example, amino acid residues that are conserved among thepolypeptides of the present invention are predicted to be particularlyunamenable to alteration.

As used herein, a “biologically active portion” of an osteoactivinprotein includes a fragment of an osteoactivin protein that can modulatebone cell differentiation or stimulate bone formation. Biologicallyactive portions of an osteoactivin protein include peptides comprisingamino acid sequences sufficiently homologous to or derived from theamino acid sequence of an osteoactivin protein, e.g., the amino acidsequence shown in SEQ ID NO:2, which include less amino acids than afull length osteoactivin proteins and which exhibit at least oneactivity of an osteoactivin protein. A biologically active portion of anosteoactivin protein can be a polypeptide which is, e.g., 10, 25, 50,100, 200 or more amino acids in length. Biologically active portions ofan osteoactivin protein can be used as targets for developing agentswhich modulate an osteoactivin-mediated activity.

Because osteoactivin proteins of this invention modulate bone celldifferentiation and bone formation, they are useful for developing noveltherapeutic compositions for bone disorders, as described in more detailbelow.

Vectors

Preferably, a biologically functional expression vector of the inventionincludes one or more regulatory sequences operatively linked to thenucleic acid sequence to be expressed. The term “regulatory sequence”includes promoters, enhancers, and other expression control elements(e.g., polyadenylation signals). Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence, as wellas tissue-specific regulatory and/or inducible sequences. The design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,and the like. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or polypeptides, includingfusion proteins or polypeptides, encoded by nucleic acids as describedherein (e.g., osteoactivin proteins, mutant forms of osteoactivinproteins, fusion proteins, and the like).

The biologically functional recombinant expression vectors of theinvention can be designed for expression of osteoactivin proteins inprokaryotic or eukaryotic cells. For example, representativeosteoactivin expression vectors are yeast expression vectors and vectorsfor expression in insect cells (e.g., a baculovirus expression vector)or a vector suitable for expression in mammalian cells such as yeast orCHO cells. Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example, using T7 promoterregulatory sequences and T7 polymerase. Suitable host cells arediscussed further in Goeddel (Meth. Enzymol. 185:3-7 (1990)).

For example, expression of proteins in prokaryotes is most often carriedout in E. coli with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionproteins. Fusion vectors add a number of amino acids to a proteinencoded therein, usually to the amino terminus of the recombinantprotein. Such fusion vectors typically serve three purposes: (1) toincrease expression of recombinant protein; (2) to increase thesolubility of the recombinant protein; and 3) to aid in the purificationof the recombinant protein by acting as a ligand in affinitypurification. Often, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the recombinant protein to enableseparation of the recombinant protein from the fusion moiety subsequentto purification of the fusion protein. Such enzymes and their cognaterecognition sequences include, but are not limited to, Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Amersham Pharmacia Biotech Inc., Piscataway, N.J.; Smith et al.,Gene 67:31-40 (1988)), and pMAL (New England Biolabs, Beverly, Mass.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A to the target recombinant protein.

Purified fusion proteins can be used in osteoactivin activity assays,(e.g., direct assays or competitive assays described in detail below),or to generate antibodies specific for osteoactivin proteins. In onenon-limiting example, a retroviral expression vector encoding a fusionprotein can be used to infect bone marrow cells which are subsequentlytransplanted into irradiated recipients. The pathology of the subjectrecipient is then examined after sufficient time has passed (e.g., sixweeks).

To maximize recombinant protein expression in E. coli, the protein canbe expressed in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., Meth.Enzymol. 185:119-128, (1990)). Another strategy is to alter thenucleotide sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (Wada et al., Nucleic AcidsRes. 20:2111-2118 (1992)). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.

When used in mammalian cells, the control functions of the expressionvector are often provided by viral regulatory elements. For example,commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus, or Simian Virus 40.

Antibodies

The present invention also encompasses antibodies that recognize andbind to the osteoactivin protein or fragment thereof (e.g., to one ormore epitopes of the protein having SEQ ID NOS:2 or 6). These antibodiesare polyclonal or monoclonal antibodies which may be provided usingstandard methods (see, e.g., Ausubel et al., supra; Coligan, J. E. etal., Current Protocols in Immunology, John Wiley & Sons, New York(1991); and Delves, P. J., Antibody Production: Essential Techniques,John Wiley & Sons, New York (1997)). Briefly, osteoactivin protein or apolypeptide fragment purified according to the methods described for anaspect of the invention are used to immunize rabbits (e.g., forpolyclonal antibodies) or mice (e.g., for monoclonal antibodies) togenerate antibody-mediated immunity to the osteoactivin protein orpolypeptide fragment used to immunize the animal. Monoclonal antibodiescan be screened by, e.g., ELISA, to identify those that show the highestaffinity for the immunizing osteoactivin protein or polypeptidefragment. The cloned cell producing the high affinity monoclonalantibody can then be propagated in vitro (where the antibody is purifiedfrom the culture supernatant) or in vivo (where the antibody is purifiedfrom ascites fluid), and can also be cryopreserved and stored frozen,e.g., at −70° C. in DMSO, to provide a potentially limitless supply ofmonoclonal antibody. Antibodies can also be provided by knownrecombinant DNA techniques.

In addition to intact monoclonal and polyclonal antibodies, theinvention also provides various fragments of an osteoactivin antibody,such as Fab, F(ab′)₂, Fv, and sFv fragments, which can be produced byproteolytic cleavage or recombinant DNA techniques. Humanized antibodiesare also provided and can be produced according to methods known in theart (see, e.g., Green et al., Nature Genetics 7:13-21 (1994)).

Also provided by the invention are osteoactivin protein-specific singlepolypeptide chain antibodies (see general methods in U.S. Pat. Nos.4,946,788 and 4,704,692); single domain antibodies (see, e.g., Ward etal., Nature 341:544-546 (1989)); and chimeric antibodies (see, e.g.,U.S. Pat. No. 4,816,567). A single-chain antibody (scFV) may beengineered by known methods (see, e.g., Colcher et al., Ann. NY Acad.Sci. 880:263-80 (1999); and Reiter, Clin. Cancer Res. 2:245-52 (1996)).The single chain antibody can be dimerized or multimerized to generatemultivalent antibodies having specificities for different epitopes ofthe same target osteoactivin protein.

Preferably, the antibody of the invention specifically binds to itsspecific ligand with a dissociation constant (K_(D)) of at least 10⁻⁵ M,more preferably, of at least 10⁻⁶ M, even more preferably, of at least10⁻⁷ M, and most preferably, with a K_(D) of at least 10⁻⁸ M.

Antibodies that specifically bind osteoactivin protein or polypeptidefragments thereof are useful, for example, in determining expressionlevels of osteoactivin protein in various tissues of the body, inWestern blotting analysis, and in immunochromatography.

A full-length osteoactivin protein or an antigenic peptide fragment ofosteoactivin can be used as an immunogen or can be used to identifyanti-osteoactivin antibodies made with other immunogens (e.g., cells,membrane preparations, and the like). The antigenic peptide ofosteoactivin preferably includes at least eight sequential amino acidresidues from SEQ ID NO:2 and encompasses an epitope of osteoactivin.Preferably, the antigenic peptide includes at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues.

Fragments of the osteoactivin amino acid residues 35-51 (SEQ ID NO:3;hereinafter “peptide 35”) or amino acid residues 551-568 (SEQ ID NO:4;hereinafter “peptide 551”), or amino acid residues 538-553 of SEQ IDNO:6 can be used, e.g., as immunogens to make or characterize thespecificity of an antibody against osteoactivin protein. Antibodiesreactive with, or specific for, any of these regions of osteoactivin, orother regions or domains described herein are provided.

Exemplary preferred epitopes encompassed by the antigenic peptide areregions of osteoactivin located on the surface of the protein, e.g.,hydrophilic regions. For example, an Emini surface probability analysisof the human osteoactivin protein sequence can be used to indicate theregions that have a particularly high probability of being localized tothe surface of the osteoactivin protein, and are thus likely toconstitute surface residues useful for targeting antibody production.However, antibodies of the invention bind an epitope on any domain orregion on osteoactivin proteins described herein.

Chimeric, humanized, but most preferably, completely human antibodiesare desirable for applications which include repeated administration,e.g., therapeutic treatment (and some diagnostic applications) of humanpatients.

Some antibodies of the invention have a reduced ability or no ability tobind an Fc receptor, for example, where it is an isotype or subtype,fragment, or other mutant, which does not support binding to an Fcreceptor, or where it has a mutagenized or deleted Fc receptor bindingregion.

An anti-osteoactivin antibody (e.g., monoclonal antibody) can be used toisolate osteoactivin by standard techniques, such as by affinitychromatography or immunoprecipitation. Moreover, an anti-osteoactivinantibody can be used to detect osteoactivin protein (e.g., in a cellularlysate or cell supernatant) in order to evaluate the abundance andpattern of expression of the protein. Anti-osteoactivin antibodies canbe used diagnostically to monitor protein levels in tissue as part of aclinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Non-limiting examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials.Non-limiting examples of suitable enzyme labels include horseradishperoxidase, alkaline phosphatase, galactosidase, andacetylcholinesterase. Non-limiting examples of suitable prosthetic groupcomplexes include streptavidinibiotin and avidinibiotin. Non-limitingexamples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin.An example of a luminescent material includes, but is not limited to,luminol. Non-limiting examples of bioluminescent materials includeluciferase, luciferin, and aequorin. Non-limiting examples of suitableradioactive material include ¹²⁵I, ¹³¹I, ³⁵S, and ³H. Coupling of labelsto antibodies can be accomplished using standard techniques (see, e.g.,Antibodies, A Laboratory Manual, Hanlos and Lane, eds. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1999).

Therapeutic Compositions

The osteoactivin-encoding nucleic acid and osteoactivin polypeptides andfragments thereof, as well as anti-osteoactivin antibodies (alsocollectively referred to herein as “active compounds”), of theinvention, or an agent that modulates osteoactivin activity orexpression, can be incorporated into therapeutic compositions. Suchcompositions typically include osteoactivin nucleic acid molecules,proteins, antibodies, or agents and preferably includes apharmaceutically acceptable delivery vehicle or carrier. As used hereinthe language “pharmaceutically acceptable delivery vehicle” includessolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. This delivery vehicle maybe targeted to the bone or bone cells by virtue of its composition, forexample, using a bisphosphanate tetracycline, or calcein. Alternatively,a vehicle may be a polymer or collagen composition that is applied tobone during surgery or by injection at the bone site (see, e.g. U.S.Pat. Nos. 4,938,763; 5,278,201; 5,324,519; 5,487,897; 5,599,552;5,702,716; 5,733,950; 5,739,176; 5,744,153; 5,759,563; 5,780,044;5,945,115; 5,990,194; and 5,631,243). Additional active compounds canalso be incorporated into the compositions.

A therapeutic composition is formulated to be compatible with itsintended route of administration. Non-limiting examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., by ingestion or inhalation), transdermal(topical), transmucosal, and rectal administration. Oral administrationor injection at a bone site is preferred. Solutions or suspensions canbe made as described in Remington's Pharmaceutical Sciences, (18^(th)ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., (1990)).

Therapeutic efficacy of such active compounds can be determined bystandard therapeutic procedures in cell cultures or experimentalanimals, e.g., for determining the ED50 (the dose therapeuticallyeffective in 50% of the population).

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage mayvary depending upon the dosage form employed and the route ofadministration. For any compound used in the method of the invention,the therapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from 0.001 to 30 mg/kgbody weight, preferably 0.01 to 25 mg/kg body weight, more preferably0.1 to 20 mg/kg body weight, and even more preferably 1 to 10 mg/kg, 2to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. Theprotein or polypeptide can be administered one time per week for between1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between3 to 7 weeks, and even more preferably for 4, 5, or 6 weeks. The skilledartisan will appreciate that certain factors may influence the dosageand timing required to effectively treat a mammal including, but notlimited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the mammal, and otherdiseases present. Moreover, treatment of a mammal with a therapeuticallyeffective amount of an osteoactivin protein, polypeptide, or antibodycan include a single treatment or, preferably, can include a series oftreatments.

For antibodies, the preferred dosage is generally 10 mg/kg to 20 mg/kgbody weight. Generally, partially humanized antibodies and fully humanantibodies have a longer half-life within the human body than otherantibodies. Accordingly, lower dosages and less frequent administrationare possible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described in Cruikshanket al. (J. Acquired Immune Deficiency Syndromes Hum. Retrovirol. 14:193(1997)).

The therapeutic compositions may also include other active or inertcomponents. Of particular interest are those mediators that promote bonegrowth or infiltration, such as cytokines and growth factors.Non-limiting exemplary mediators for this purpose include interleukin-1,tumor necrosis factor, lymphotoxin, interleukin-6, prostaglandins of theE-series, leukotrienes, lipopolysaccharides, transforming growthfactor-β, and colony-stimulating factors. Agents that promote bonegrowth, such as bone morphogenic proteins, are also useful.

Within the present invention, a “therapeutically effective amount” of atherapeutic composition is that amount which produces a desired effect.For example, a therapeutically effective amount is the amount of thetherapeutic composition comprising the active osteoactivin proteincompound herein required to provide an effect in reversing the symptomsof the bone disorder. Such therapeutically effective amounts will bedetermined using routine optimization techniques that are dependent onthe particular condition to be treated, the condition of the patient,the route of administration, the formulation, and the judgment of thepractitioner and other factors evident to those skilled in the art. Thedosage required for the therapeutic compositions of the invention, forexample, in osteoporosis where an increase in bone formation is desired,is manifested as a statistically significant difference in bone massbetween treatment and control groups. Other measurements of increases inbone formation and/or bone healing may include, for example, tests forbreaking strength and tension, breaking strength and torsion, 4-pointbending, increased connectivity in bone biopsies and other biomechanicaltests well known to those skilled in the art. General guidance fortreatment regimens is obtained from experiments carried out in animalmodels.

Therapeutic Methods

The therapeutic compositions of the present invention may be used tomodulate the differentiation of osteoblasts, osteoblast precursors, andmesenchymal cells in vivo, in vitro, or ex vivo, and to modulate bonecell differentiation and bone formation. As used herein, the term“osteoblast precursor” refers to a cell that is committed to adifferentiation pathway, but that generally does not express markers orfunction as a mature, fully differentiated cell. Such cells include“mesenchymal cells” or “mesenchymal stem cells,” which are pluripotentcells that are capable of dividing many times and whose progeny willgive rise to skeletal tissues, including cartilage, bone (osteogeniccells), tendon, ligament, marrow stroma and connective tissue. Thedisclosed therapeutic compositions or methods are useful for stimulatingosteogenesis.

In a preferred method, bone cell differentiation and bone formation isstimulated by administering to a mammal a therapeutic compositioncomprising a nucleic acid molecule encoding an osteoactivin protein, orcomprising an osteoactivin protein or biologically active polypeptidefragment thereof, or comprising an agent that stimulates osteoactivinexpression or activity. The appropriate agent can be determined based onscreening assays described herein. Bone disorders in mammals that may betreated or prevented by administering one of the above-describedtherapeutic compositions of the invention include those diseases orpathological conditions in which stimulation of bone cell formation isdesired, such as with osteoporosis or bone trauma.

As an alternative, bone cell differentiation and bone formation can beinhibited by administering to a mammal a therapeutic compositioncomprising an osteoactivin antibody, an osteoactivin antisense nucleicacid, or an agent that inhibits osteoactivin expression or activity.Such therapy is used in treating, for example, osteopetrosis. Examplesof osteoactivin antibodies include, for example, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab,F(ab′)₂ and Fab expression library fragments, scFV molecules, andepitope-binding fragments thereof. An antisense oligonucleotide directedto the osteoactivin gene or mRNA to inhibit its expression is madeaccording to standard techniques. (See, e.g., Agrawal et al. Methods inMolecular Biology: Protocols for Oligonucelotides and Analogs. Vol. 20(1993)). An agent that inhibits osteoactivin activity or expression isidentified by screening assays, as described herein.

In another preferred method, hematopoietic osteogenic cells are removedex vivo from the cell population, either before or after contact orstimulation with a disclosed therapeutic composition. Through well-knownpractices, the osteogenic cells may be expanded. The expanded osteogeniccells can be infused or reinfused into a mammal in need thereof.

Screening Methods

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators of osteoactivin expression and orosteoactivin activity. Such modulators (i.e., candidates, testcompounds, agents, proteins, peptides, peptidomimetics, peptoids, smallmolecules or other chemical entities) stimulate or inhibit osteoactivinexpression or osteoactivin activity. Therefore, agents thus identifiedcan be used to regulate bone cell differentiation and bone formation ina therapeutic protocol.

The test compounds used for screening may be selected individually orobtained from a compound library. Such libraries include biologicallibraries, peptoid libraries (libraries of molecules having thefunctionalities of peptides, but with a novel, non-peptide backbonewhich are resistant to enzymatic degradation but which neverthelessremain bioactive)(see, e.g., Zuckermann, J. Med. Chem. 37:2678-85(1994)), spatially addressable parallel solid phase or solution phaselibraries, synthetic library methods requiring deconvolution, the“one-bead one-compound” library method, and synthetic library methodsusing affinity chromatography selection. The biological library andpeptoid library approaches are limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, Anticancer Drug Dis. 12:145(1997)).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example, in: DeWitt et al., Proc. Natl. Acad. Sci.(USA) 90:6909 (1993); Erb et al., Proc. Natl. Acad. Sci. (USA) 91:11422(1994); Zuckermann et al., J. Med. Chem., 37:2678 (1994); Cho et al.,Science, 261:1303 (1995); Carrell et al., Angew. Chem. Int. Ed. Engl.33:2059 (1994); Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061(1994); and in Gallop et al., J. Med. Chem. 37:1233 (1994).

Libraries of compounds may be presented in solution (e.g., Houghten,Biotechniques, 13:412-421 (1992)), or on beads (Lam, Nature 354:82-841(1991)), on chips (Fodor, Nature 364:555-556 (1993)), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409),plasmids (Cull et al., Proc. Natl. Acad. Sci. USA., 89:1865-1869 (1992))or on phage (Scott et al., Science 249:386-390 (1990); Devlin, Science249:404-406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. (USA)87:6378-6382 (1990); Felici, J. Mol. Biol. 222:301-310 (1991); Ladnersupra.).

In one embodiment, the invention provides an assay for identifying anagent that modulates the expression of an osteoactivin protein. Themethod uses cells capable of expressing a gene under the control of theregulatory element(s) of an osteoactivin gene. Such cells include thosewhich are capable of expressing an endogenous osteoactivin gene (e.g.,an osteoblast cell line) or a cell transfected with a transgenecomprising an osteoactivin regulatory element (e.g., an osteoactivinpromoter) fused to a nucleic acid sequence encoding a polypeptide (e.g.,an osteoactivin protein or a reporter protein), such that theosteoactivin gene regulatory element controls expression of the codingsequence.

In a preferred embodiment, the human osteoactivin gene promoter is usedto identify agents that modulate osteoactivin gene expression. Thesequence of the human osteoactivin promoter can be found in cloneRG271G13 from the Genome Sequencing Center (Washington Univ., St. Louis,Mo.)(GenBank Accession Number AC005082) which encodes the humanosteoactivin gene and promoter. The initiation of translation (ATG)starts at bp 86355 of this clone and a probable TTATAA box starts at bp86510. A portion of this promoter, preferably 8-10 kb upstream of theATG start site, is cloned into a vector containing, for example, areporter gene (such as secreted alkaline phosphatase, green fluorescentprotein, or luciferase) and a gene encoding antibiotic resistance. Thevector is transfected into an osteoblastic cell line, such as, e.g.,MG-63, HO5, or SaOS, (all available from the American Type CultureCollection, Manassas, Va.) which expresses an endogenous osteoactivingene. Following transfection, cells that incorporate the construct areselected by their ability to grow in the presence of an appropriateantibiotic and, subsequently, clonal cell lines are established bylimiting dilution.

The expression of the reporter gene in these cell lines is determined bythe appropriate measurement of reporter gene expression. Expression ismeasured at confluence and compared to an appropriate control cell line,such as a cell line transfected with a construct in which the promoterwas cloned in the opposite orientation, in a cell line transfected witha construct in which the promoter was absent, and in a different cellline which does not express endogenous osteoactivin and which istransfected with the promoter-reporter construct.

Once a bona fide clonal osteoblastic cell line expressing the humanosteoactivin promoter-reporter construct is identified, the cell line isexpanded and submitted for high throughput screening to identify agentscapable of modulating (e.g., increasing or decreasing) the expression ofthe reporter gene. When reporter expression is greater in the presenceof the candidate compound than in its absence, the candidate compound isidentified as a stimulator of osteoactivin expression. Alternatively,when reporter expression is less in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of osteoactivin expression. The level of reporter expressioncan be determined by methods described herein for detecting the level ofreporter osteoactivin mRNA or protein produced by the cell.

In another embodiment, the osteoactivin protein, or biologically activeportion thereof, is contacted with a compound known to bindosteoactivin, e.g., an osteoactivin antibody, to form an assay mixture.The assay mixture is then contacted with a test compound, and theability of the test compound to interact with an osteoactivin protein isdetermined. Determining the ability of the test compound to interactwith an osteoactivin protein includes determining the ability of thetest compound to preferentially bind to osteoactivin or biologicallyactive portion thereof, or to modulate the activity of osteoactivin, ascompared to the known compound.

Additionally, the invention encompasses diagnostic and prognosticassays. Accordingly, the presence, level, or absence of osteoactivinprotein or nucleic acid expression in a biological sample can beevaluated by methods described herein. In one embodiment, the methodcomprises obtaining a biological sample from a test mammal andcontacting the biological sample with a compound or an agent capable ofdetecting osteoactivin protein or nucleic acid (e.g., mRNA, genomic DNA)that encodes osteoactivin protein such that the presence of osteoactivinprotein or nucleic acid is detected in the biological sample. The term“biological sample” includes tissues, cells and biological fluidsisolated from a mammal, as well as tissues, cells and fluids presentwithin a mammal. A preferred biological sample is serum. The level ofexpression of the osteoactivin gene can be measured in a number of waysincluding, but not limited to, measuring the mRNA encoded by theosteoactivin gene; measuring the amount of protein encoded by theosteoactivin gene; or measuring the activity of the protein encoded bythe osteoactivin gene.

In another example, a control sample is contacted with a compound oragent capable of detecting osteoactivin mRNA, or genomic DNA. Thepresence of osteoactivin mRNA or genomic DNA in the control sample isthen compared with the presence or level of osteoactivin mRNA or genomicDNA in the test sample. The control sample is obtained from a normalbone, whereas the test sample can be obtained from a site of aberrantbone growth.

A variety of methods can be used to determine the level of osteoactivinprotein expressed. In general, these methods include using an agent thatselectively binds to osteoactivin, such as an antibody, to evaluate thelevel of osteoactivin in the sample. In a preferred embodiment, theantibody bears a detectable label. Useful antibodies include any ofthose described above.

The detection methods can be used to detect osteoactivin protein in abiological sample in vitro as well as in vivo. In vitro techniques fordetection of osteoactivin protein include enzyme linked immunosorbentassays (ELISAs), immunoprecipitations, immunofluorescence, enzymeimmunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. Invivo techniques for detection of osteoactivin protein includeintroducing into a mammal a labeled anti-osteoactivin antibody. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a mammal can be detected by standard imagingtechniques.

The invention is further illustrated by the following examples thatshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES

Reagents

All chemicals were of molecular biology grade or higher and werepurchased from Sigma (St. Louis, Mo.) unless otherwise noted. All cellculture media were purchased from Life Technologies (Gaithersburg, Md.).

Animals

An inbred colony of osteopetrotic (op) mutant rats consisting ofheterozygous breeders is maintained at Temple University School ofMedicine (Philadelphia, Pa.). Homozygous mutants (op/op) aredistinguished from normal littermates (+/?) by radiographic analysisbetween 1 and 3 days after birth by the failure of the mutants todevelop bone marrow cavities. Because the genotype of phenotypicallynormal rats cannot be distinguished, except by breeding experiments, thenormal littermates used in this study were of either heterozygous (+/op)or homozygous (+/+) normal genotype. All animals were maintained andused according to the principles in the NIH Guide for the Care and Useof Laboratory Animals (1985), and guidelines established by the IACUC ofTemple University.

Example 1 Primary Osteoblast Cultures

Normal diploid osteoblasts were isolated from the calvaria of 1-3 dayold op/op mutant or normal littermates rats by sequentialtrypsin/collagenase digestion and plated in 100 mm dishes in minimumessential medium (MEM) supplemented with 10% fetal bovine serum (FBS;Gemini Bioproducts, Calabases, Calif.) at a density of 5×10⁵ cells/dish(Owen, et al., J. Cell. Physiol. 143:420-430 (1990)). Media was changedevery other day throughout the time course of culture and for mediachanges after day 6 of culture, MEMα supplemented with 50 μg/ml ascorbicacid, 2 mM inorganic phosphate, and 10% FBS was used to feed the cells.

Example 2 RNA Isolation

Total cellular RNA was isolated from calvaria and long bones (femurs andtibias) harvested from two week old op/op mutant rats and their normallittermates.

Prior to freezing, the ends of the long bones were removed at the growthplate and bone marrow was flushed from the shafts of normal bones withsaline (4° C.) using a 25-gauge needle. Flushing of the bone marrow wasonly possible in normal rats; op mutants had no marrow cavities. TotalRNA was prepared from pools of a minimum of six samples per phenotypeand bone site (calvaria versus long bone). Total RNA was prepared asdescribed by Thiede et al. (Endocrinology 135:929-37 (1994)). Briefly,samples were frozen in liquid nitrogen, pulverized on dry ice, andhomogenized in 5 M guanidinium isothiocyanate, 72 mM β-mercaptoethanol,and 0.5% sarkosyl. Homogenates were layered over a cesium chloride(CsCl) cushion (5.7 M CsCl and 30 mM sodium acetate (NaAc)), centrifugedat 100,000×g overnight, and total RNA recovered by precipitation of theresulting pellets.

RNA was isolated from the rat osteoblast cultures, as well as fromliver, spleen, thymus and brain harvested from 2, 4, and 6 week-old oprats and their normal littermates using TRIzol® (Life Technologies,Gaithersburg, Md.). The RNA concentration of each sample was quantitatedby absorbance at 260 nm. The integrity and accuracy of thespectrophotometric measurement of each RNA sample was assessed byelectrophoresis of 1 μg on an ethidium bromide-stained,formaldehyde-agarose minigel.

Example 3 Differential Display of mRNA

Prior to differential display, bone RNA samples were treated withDNase.I (Roche Molecular Biochemicals, Indianapolis, Ind.) to eliminateany potential contamination with genomic DNA. The basic principle ofmRNA differential display was first described by Liang and Pardee (1992)Science 257:967-971. Briefly, 0.5 μg RNA from each sample (total of 4independent samples, mutant and normal, calvaria and long bone) wasreverse transcribed using each of 12 two-base-anchored oligo-dT primersprovided in the Hieroglyph mRNA profile kits (Beckman Coulter Inc.,Fullerton, Calif.) to subdivide the mRNA population. First strand cDNAswere amplified by the polymerase chain reaction (PCR) for 30 cyclesusing one of 4 upstream arbitrary primers (also provided in the kit) andthe same anchoring primers used for first strand synthesis. Thisresulted in 48 possible primer combinations for each kit (total of 5kits) and each PCR amplification was run in duplicate from the samefirst-strand cDNA template. All amplified cDNAs were radiolabeled with³³P-dATP ([α-³³P]dATP, 2500 Ci/mmol, Amersham Pharmacia Biotech,Piscataway, N.J.). The radiolabeled PCR products were electrophoresed on4.5% denaturing polyacrylamide gels and dried using the Genomyx LRdifferential display apparatus (Beckman Coulter).

As shown in FIG. 3, differential display analysis of op/op mutant versusnormal calvaria and long bone RNA revealed an mRNA that wasoverexpressed in the op/op mutant bone RNA.

Following autoradiography, the bands were visually assessed and thoserepresenting differentially expressed cDNAs (exclusively expressed orhighly overexpressed in one phenotype and confirmed in duplicate PCRamplifications) were excised from the gel. Each cDNA of interest wasreamplified by PCR and used to probe a Northern blot to confirm itsdifferential expression.

Example 4 Northern Blot Analysis

Twenty μg of total RNA from op mutant and normal bone/soft tissue ornormal osteoblast cultures were electrophoresed on 1%formaldehyde-agarose gels and transferred to nylon membranes (Scheicher& Schuell, Keene, N.H.). Blots were hybridized with a ³²P-labeled([α-³²P]dCTP, 6000 Ci/mmol, Amersham Pharmacia Biotech, Piscataway,N.J.) full length rat osteoactivin cDNA probe (Rediprime™II, AmershamPharmacia Biotech) using methods described in Thiede et al.,Endocrinology 135:929-37 (1994). Blots were then autoradiographed,stripped and re-probed with an 18s rRNA probe used as a control tonormalize for differences in loading and transfer.

FIG. 4A shows that the osteoactivin mRNA expression levels were 5-7times higher in the op mutant (M) calvaria as compared to the normal (N)calvaria. cDNAs which were confirmed to be differentially expressed byNorthern blot analysis were cloned into PCR-Script (Stratagene, Lajolla,Calif.), miniprep DNA was prepared, and plasmids with the appropriatelysized inserts were sequenced.

Example 5 Cloning of Rat Osteoactivin cDNA

Approximately 600 bp of sequence corresponding to the 3′ end of ratosteoactivin was obtained from the differential display clone. Thisfragment was used as a probe to screen a rat kidney cDNA library byconventional means. A single clone was identified after three rounds ofscreening and DNA sequence analysis showed that it contained an openreading frame of 1719 bp as depicted in FIG. 1A. Following confirmationby Northern blot analysis (see FIGS. 4A and 4B), this clone wassequenced and found to be related to a human sequence (nmb) of unknownfunction. This cDNA and predicted protein was named osteoactivin toreflect its potential role in osteoblast function. Cloning of the entirecoding region of osteoactivin nucleic acid sequence revealed an openreading frame capable of encoding a protein of 572 amino acids with 70%identity to human nmb as depicted in FIG. 2B. Osteoactivin has apredicted molecular weight of 63.8 kD. Hydropathy analysis, as providedin FIG. 1B revealed a potential leader sequence and several potentialtransmembrane spanning domains throughout the protein, suggestingmembrane association.

Example 6 DNA Sequencing

DNA was sequenced using standard dideoxy methodologies. Gaps andambiguities in the sequence were handled by direct sequencing ofrequired regions using specific primers. The nucleic acid sequence ofrat osteoactivin has been deposited in GenBank under Accession NumberAF184983.

In FIGS. 2A and 2B, the nucleotide (SEQ ID NOS: 1, 7, and 8) andpredicted amino acid sequences (SEQ ID NOS: 2, 5, and 6), respectively,of rat osteoactivin and human and mouse nmb were compared. FIG. 2Areveals that there is a 76% sequence identity in the nucleotidesequences between rat and human. The predicted protein sequence of ratosteoactivin has a proline serine-rich 14 amino acid insertion beginningat residue 333 that is not present in the human nmb protein sequence, asshown in FIG. 2B. On the protein level, the sequences of ratosteoactivin and human nmb are 69% identical.

Example 7 Antibody Preparation

Peptide 35 H-CPDHMRENNQLRGWSSDE-NH₂ (SEQ ID NO:3) and peptide 551H-KAPFSRGDREKDPLLQDKC-NH₂ (SEQ ID NO:4) were conjugated to KeyholeLimpet Hemocyanin (KLH) by Cys residues added at N-terminal end ofpeptide 35 of the osteoactivin protein (SEQ ID NO:2) or at C-terminalend of peptide 551. For each peptide, two chickens were immunized with100 μg/chicken/Freunds complete adjuvant on the following immunizationschedule: Day 1, pre-immune eggs collected, first boost; Day 14, 2^(nd)boost; Day 28, 3^(rd) boost; Day 42, 4^(th) boost; and Day 49, begincollecting eggs. Twelve eggs were collected and pooled from eachchicken. Affinity purified peptide-specific immune IgY was purified byaffinity chromatography using peptide immobilized on column. (Hanlos andLane, supra).

Example 8 Immunolocalization of Osteoactivin in Primary Rat Osteoblasts

Primary osteoblasts isolated from newborn normal rat calvaria wereplated in 100 mm dishes at a density of 5×10⁵ cells/dish in Earl'smedium supplemented with 10% fetal bovine serum (FBS), 50 μg/ml ascorbicacid, and 10 mM β-glycerolphosphate and cultured for 1 week. Cells werefixed in 4% paraformaldehyde for 10 minutes at 4° C., treated with 0.1%Triton X-100 for 10 minutes at room temperature (RT), and then blockedwith 10% goat serum for 15 minutes at RT. They were then stained with ananti-osteoactivin polyclonal antibody raised in chickens against aminoacid residues 551-558 at the C-terminal end of the osteoactivin proteinor with buffer alone at a dilution of 1:50 in phosphate buffered saline(PBS) and incubated overnight at 4° C. Following three washes in PBS (5minutes each), Cy3 conjugated goat anti-chicken secondary antibody(Jackson Immuno Research, West Grove, Pa.), which served as afluorescent marker, was added (1:1000 in PBS) and incubated for 1 hr atRT. Osteoactivin was visualized by fluorescence microscopy (FIG. 5).

In another experiment to further study the properties of the protein,immunofluorescent staining was performed on primary rat osteoblastscultured in chamber slides for 5 days following plating at a density of12,400 cells per chamber. The cells were fixed in 4% paraformaldehydeand were stained overnight at 4° C. using 1 μg/ml polyclonalosteoactivin antibody (Cambridge Research Biochemicals, Billingham,Cleveland, UK) as the primary antibody. The antibody was raised inchickens against amino acid residues 551-568 at the C-terminal end ofthe rat osteoactivin protein. More specifically, the polyclonal antibodywas made against the following peptide conjugated to keyhole limpethemocyanin: H-KAPFSRGDREKDPLLQDKC-NH₂. (SEQ ID NO: 4). The primaryantibody was then detected following washing by incubation with a1:10,000 dilution of a Cy3-conjugated donkey anti-chicken secondaryantibody (Jackson ImmunoResearch, West Grove, Pa.) for 1 hr at roomtemperature. The results are shown in FIG. 5A, which indicatesimmunofluorescent staining in the perinuclear region of the cell.

For co-localization within the RER, cells were stained with the cellpermanent, fluorescent probe DiOC₅, a membrane marker for RER (MolecularProbes, Eugene, Oreg.) following incubation with the primary andsecondary antibodies. The results, shown in FIG. 5B and the overlay ofFIGS. 5A and 5B in FIG. 5C, indicate that osteoactivin co-localizes inthe RER in primary rat osteoblasts, suggesting that osteoactivin is asecreted protein.

Example 9 Northern Blot Analysis of Osteoactivin Expression in Calvariaand Long Bone of Rats of Different Ages

Twenty μg of total RNA isolated from normal (N) or mutant (M) calvariaor long bones at 2, 4, and 6 weeks of age was electrophoresed in a 1%agarose formaldehyde gel, blotted, and probed for osteoactivin, asdescribed in Example 4 above. Northern analysis was repeated three timesusing independent RNA samples with similar results.

As shown in FIG. 6, osteoactivin expression was higher in mutantcalvaria and long bones when compared to normal bones at all agesexamined. In normal animals, osteoactivin expression decreases with agein both calvaria and long bones. However, in the mutants, osteoactivinexpression is still detectable in the calvaria at six weeks of age and,in long bones, is highly expressed at all ages examined.

Example 10 Northern Blot Analysis of Osteoactivin Expression in PrimaryRat Osteoblast Cultures

Total RNA was isolated from normal (N) or mutant (M) osteoblast culturesat 1, 2 and 3 weeks of culture. Twenty μg of RNA from each sample wassubjected to Northern blot analysis, as described in Example 4 above.

As shown in FIG. 7A, the temporal pattern demonstrated a remarkableincrease in expression between 1 week (proliferation stage) and 2 weeks(matrix maturation stage), with a modest decrease at 3 weeks in culture(mineralization stage). There was no significant difference betweenosteoblast cultures derived from normal or mutant calvaria. The blot wasstripped and re-probed for 18s rRNA to normalize for differences inloading and/or blotting (FIG. 7B). Similar results were obtained fromtwo separate experiments.

Example 11 Detection of Secreted Osteoactivin Protein in OsteoblastCulture Conditioned Media

Osteoblast cultures were established from the calvaria of newborn op/opmutant rats or their normal littermates by established methods andplated at a density of 500,000 cells per 100 mm dish in MEMαsupplemented with 10% FBS. Cell culture media was changed every otherday. Six days after the initiation of the cultures, the cells werewashed twice with culture media lacking FBS. 10 ml of serum free culturemedium was then added per 100 mm dish and the cells were cultured for anadditional 24 hours. This medium was then harvested and the proteinsconcentrated using a 10 kD molecular weight cut-off concentrator(Millipore, Bedford, Mass.). The concentration of protein in theseconcentrated samples was determined by the method of Bradford (Analyst.Biochem. 72:246-54 (1976)) using reagents from Pierce (Rockford, Ill.).10 μg of concentrated protein from the op/op mutant or normal osteoblastcultures was electrophoresed in a 10% polyacrylamide-sodium dodecylsulfate (SDS) gel and then transferred to polyvinylidene fluoride (PVDF)membrane by electroblotting. This blot was blocked for 1 hour at roomtemperature in 10% non-fat milk in phosphate buffered saline (PBS)-0.2%Tween 20 (PBS-Tween) and then incubated overnight at 4° C. in blockingsolution containing 0.325 μg/ml chicken anti-rat osteoactivin 551antibody. Following four washes in PBS-Tween, the blot was incubated atRT for 1 hour in blocking solution containing a 1:5000 dilution ofhorseradish peroxidase conjugated donkey anti-chicken antibody (JacksonImmunoresearch, West Grove, Pa.). Following four washes in PBS-Tween,the blot was developed using Enhanced Chemiluminescence (ECL) reagents(Amersham Pharmacia Biotech, Piscataway, N.J.) and visualized byexposure to film.

The results shown in FIG. 8 demonstrate that one week old mutantosteoblast cultures secrete significantly more osteoactivin protein thannormal osteoblast cultures.

Example 12 Detection of Osteoactivin Protein in the Tibia of Normal andop/op Mutant Rats

Tibia were harvested from op/op mutant rats or from their normallittermates at the indicated weeks of age, immediately frozen in liquidnitrogen, and stored at −80° C. until all samples were collected. Thefrozen bones were then homogenized in RIPA buffer (50 mM Tris pH 7.5,150 mM NaCl, 1.0% NP-40, 0.1% SDS, 0.5% sodium deoxycholate; MolecularCloning: A Laboratorv Manual, Cold Spring Harbor Press (1989), 18.38)containing protease inhibitors (Complete tablets, Roche MolecularBiochemicals, Indianapolis, Ind.), and the insoluble material removed bycentrifugation. The concentration of soluble proteins was determined bythe method of Bradford (Analyst. Biochem. 72:246-54 (1976)) usingreagents from Pierce (Rockford, Ill.). 40 μg of protein from the op/opmutant or normal tibia was electrophoresed in a 10% polyacrylamide-SDSgel and then transferred to PVDF membranes by electroblotting. This blotblocked for 1 hr at room temperature in 10% non-fat milk in PBS-Tweenand then incubated overnight 4° C. in blocking solution containing 0.325μg/ml chicken anti-rat osteoactivin 551 antibody. Following four washesin PBS-Tween, the blot was incubated at RT for 1 hour in blockingsolution containing a 1:5000 dilution of horseradish peroxidaseconjugated donkey anti-chicken antibody (Jackson Immunoresearch, WestGrove, Pa.). Following four washes in PBS-Tween, the blot was developedusing ECL reagents (Pierce (Rockford, Ill.) and visualized by exposureto film. The blot was then stripped of the antibodies and detectionreagents and blocked as described. The blot was incubated overnight at4° C. in blocking solution containing 0.75 μg/ml mouse anti-rat tubulinantibody as a control for protein loading between the samples and forthe efficiency of blotting across the gel. Following four washes inPBS-Tween, the blot was incubated at RT for 1 hour in blocking solutioncontaining a 1:5000 dilution of horseradish peroxidase conjugated donkeyanti-rabbit antibody (Jackson Immunoresearch, West Grove, Pa.), washed,developed, and visualized as described above. The film images from theosteoactivin and β-tubulin experiments were quantitated by scanningdensitometry and the data were expressed as a ratio of the osteoactivinto β-tubulin signal in each sample (FIG. 9).

These results show that the expression of osteoactivin protein wassignificantly higher in mutant versus normal rat tibia in all agesexamined.

Example 13 Inhibition of Osteoblast Differentiation Following Treatmentwith Antibodies against Osteoactivin

Osteoblast cultures were established from the calvaria of newborn op/opmutant rats or their normal littermates by established methods andplated at a density of 14,200 cells per well of a 24-well plate in MEMαsupplemented with 10% fetal bovine serum. Cell culture media was changedevery other day and, beginning at day 6 of culture, cells were fed withdifferentiation medium (MEMα supplemented with 10% FBS, 50 μg/mlascorbic acid, 10 mM β-glycerophosphate). Beginning at the media changeon day 2 of culture and for every subsequent media change, affinitypurified antibodies to rat osteoactivin (antibody 551) or controlnon-immune IgY antibodies were added to the fresh culture medium finalconcentrations of 4, 20, or 40 μg/ml. All cultures were terminated at 21days and analyzed for calcium deposition in the cell/matrix layer usinga colorimetric kit from Sigma.

These data (FIG. 10) indicate that the antibodies to osteoactivininhibit rat osteoblast differentiation in vitro (as measured by calciumdeposition) in a dose dependent manner.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for stimulating bone cell differentiation or bone formationat a bone site in a mammal, the method comprising directly administeringto the bone site in the mammal a bone-stimulatory amount of a nucleicacid molecule encoding an osteoactivin protein operably linked to anexpression control sequence, wherein the encoded osteoactivin proteinstimulates the bone cell differentiation or bone formation at the bonesite.
 2. The method of claim 1, wherein said mammal is a human.
 3. Themethod of claim 1, wherein said osteoactivin protein is a humanosteoactivin protein.
 4. The method of claim 3, wherein saidosteoactivin protein comprises the amino acid of SEQ ID NO: 6 or theamino acids 23-560 of SEQ ID NO:
 6. 5. The method of claim 1, whereinsaid expression control sequence comprises a tissue-specific regulatorysequence or an inducible regulatory sequence.
 6. The method of claim 1,wherein said expression control sequence directs constitutive expressionof the nucleic acid molecule encoding the osteoactivin protein.
 7. Amethod for stimulating bone cell differentiation or bone formation at abone site in a mammal, the method comprising (a) extracting osteoblastsfrom said mammal; (b) introducing into said osteoblasts a nucleic acidencoding an osteoactivin protein operably linked to an expressioncontrol sequence, wherein said osteoblasts express the encodedosteoactivin protein; and (c) directly administering the transformedosteoblasts of step (b) to the bone site in said mammal, wherein thetransformed osteoblasts of step (b) stimulate the bone celldifferentiation or bone formation at the bone site.
 8. The method ofclaim 7, wherein said mammal is a human.
 9. The method of claim 7,wherein said osteoactivin protein is a human osteoactivin protein. 10.The method of claim 9, wherein said osteoactivin protein comprises theamino acid of SEQ ID NO: 6 or the amino acids 23-560 of SEQ ID NO: 6.11. The method of claim 7, wherein said expression control sequencecomprises a tissue-specific regulatory sequence or an inducibleregulatory sequence.
 12. The method of claim 7, wherein said expressioncontrol sequence directs the constitutive expression of the nucleic acidmolecule encoding the osteoactivin protein.
 13. An isolated osteoblastcomprising a transgene encoding an osteoactivin protein operably linkedto an expression control sequence.
 14. The osteoblast of claim 13,wherein said osteoblast is obtained from a mammal.
 15. The osteoblast ofclaim 14, wherein said mammal is a human.
 16. The osteoblast of claim13, wherein said osteoactivin protein is a human osteoactivin protein.17. The method of claim 16, wherein said osteoactivin protein comprisesthe amino acid of SEQ ID NO: 6 or the amino acids 23-560 of SEQ ID NO:6.
 18. The osteoblast of claim 13, wherein said expression controlsequence comprises a tissue-specific regulatory sequence or an inducibleregulatory sequence.
 19. The osteoblast of claim 13, wherein saidexpression control sequence directs the constitutive expression of thenucleic acid molecule encoding the osteoactivin protein.